Within the last decade, the emergence of multi-drug resistant pathogens has led to the increased use of vaccines as a method of treating infectious disease. New vaccines are being developed at a rapid pace, and assays to determine the in vivo effectiveness of these vaccines are also being developed. Several assays have been developed that determine the effectiveness of vaccines by detecting vaccine-specific antibodies generated in vivo after vaccine administration. These assays include radioantigen binding assays and enzyme-linked immunosorbant assays (ELISAs) (Schiffman et al., J. Immunol. Meth. 33:133-44, 1980; Nahm et al., J. Infect. Dis. 173:113-8, 1996; Quataert et al., Clin. Diagn. Lab. Immunol. 2:590-7, 1995). One problem with these assays is that they measure the total amount of antibody generated in response to administration of the vaccine, without regard to whether the antibodies actually provide a protective response in the immunized individual. Therefore, total antibody assays do not provide an accurate estimation as to the in vivo effectiveness of a vaccine.
Whether a vaccine provides protective immunity or simply generates an antibody response depends upon the type of infection the vaccine seeks to prevent as well as the type of antibodies generated in response to administration of the vaccine. For example, a protective immune response to pathogens such as Streptococcus pneumoniae involves opsonophagocytosis of the infectious agents. Opsonophagocytosis is the binding (or opsonization) of antibodies and complement (or complement components) to the infectious agent, and the subsequent uptake of the infectious agent by effector cells via the binding of the effector cells to the antibody/antigen complex. Therefore, a protective immune response against such pathogens includes more than the mere generation of antibodies that bind the pathogen. A protective immune response includes the generation of “functional” antibodies that bind to the infectious agents and also provide a means for uptake and clearance by effector cells. Another aspect of functionality is the ability to interact with complement reactions that may be involved in opsonophagocytosis.
One functional assay that has been developed to measure the effectiveness of vaccines is the opsonophagocytic assay. Opsonophagocytic assays more closely resemble animal models, and appear to provide a closer correlation with serotype-specific vaccine effectiveness than other prior art assays such as ELISAs (Wenger et al., Laboratory correlates for protective effectiveness of pneumococcal vaccines: how can they be identified and validated? Abstr. G37 in Program and abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, 1996). Measurement of the effectiveness of a vaccine designed to provide protection against infection by multiple serotypes of a pathogen involves measurement of multiple functional antibodies generated toward each different serotype. Although the functional assays described above can be run multiple times for each different serotype present in the vaccine, such a practice can be time consuming, labor intensive, and require a large quantity of serum for evaluation. These difficulties can be partially alleviated by evaluating multiple serotypes simultaneously (for example see Bogaert et al., Vaccine, 22:4014-20, 2004; Kim and Nahm, Clin. Diag. Lab. Immunol. 10:616-21, 2003; and Martinez et al., Clin. Diag. Lab. Immunol. 6:581-586, 1999). However, the method of Martinez et al. (Clin. Diag. Lab. Immunol. 6:581-586, 1999) can be limited by the number of fluorophores that can be simultaneously detected by the flow cytometer, and the methods of Bogaert et al. (Vaccine, 22:4014-20, 2004) and Kim and Nahm (Clin. Diag. Lab. Immunol. 10:616-21, 2003) involve waiting for growth of bacterial colonies, and then counting the colonies, which can be time consuming.
Therefore, a rapid, automated, assay is needed, particularly one that can simultaneously measure functional antibodies directed against multiple serotypes of pathogens in a single day.